This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The hydroxylation of unactivated C-H bond is a transformation of great fundamental and practical interest. A well known class of enzymes capable of this transformation is the bacterial multicomponent mooxygenases (BMMs)family utilizing four-helix bundle protein folds (alpha4) to provide 2 His and 4 carboxylate ligands to their diiron active sites [1]. We are currently investigating two novel diiron enzymes, deoxyhypusine hydroxylase (DOHH) and CmlA, that effect this transformation. Although crystal structures of these two enzymes have not been obtained, sequence analysis indicates that both DOHH [2] and CmlA [3] remarkably deviate from the highly conserved structural motif of BMMs. In addition, unlike BMMs, DOHH and CmlA only react with protein-bound substrates and their hydroxylation is strictly stereospecific.